The present invention relates to an angiogenesis inhibitor comprising a cysteine protease inhibitory compound.
An angiogenesis is a phenomenon wherein new blood vessels are created to form a new vascular network in the living body. The angiogenesis is found under normal physiological environment such as genesis and reproduction with regard to embryo, fetus, placenta, uterus and the like. It is also a pathologic phenomenon which accompanies wound healing, inflammation, growth of tumor and the like, and which is ophthalmologically seen in diabetic retinopathy, prematurity retinopathy, retinal venous occlusion, senile discoid macular degeneration and the like.
The angiogenesis greatly varies depending on the function and growth of endothelial cells, and is considered to be a cascade reaction which proceeds in the smallest vein along the following steps. That is, new blood vessels are presumably formed as a result of consecutive elementary reactions of (1) activation of vascular endothelial cells which are in the stage of rest upon differentiation, (2) destruction of cell matrix such as basement membrane by endothelial cells which expressed protease activity, (3) migration of endothelial cells, (4) proliferation of endothelial cells and (5) tube-formation by differentiation of endothelial cells [T. Oikawa, Drug News Perspest, Vol. 6, pp. 157-162 (1993)]. Each step of these reactions has been clarified to be promoted by angiogenesis promoters. Such angiogenesis promoters include, for example, blood vessel inducing factors [e.g., tumor angiogenetic factor (TAF)] secreted from tumor tissues, and growth factors such as fibroblast growth factor (FGF) present in various normal tissues, endothelial cell growth facor derived from platelets and vascular endothelial cell growth factor. In addition, cytokine, prostaglandine, monobutylin and angiogenine reportedly have similar direct or indirect effects [M. Klagsbrun et al., Annu. Rev. Physiol., Vol. 53, pp. 217-239 (1991)].
A substance which suppresses such angiogenesis include angiostatic steroids [Folkman, J. et al., Science, Vol. 221, p. 719 (1983)] such as cortisone which inhibits growth of endothelial cells; medroxyproge-sterone acetate which inhibits production of plasminogen activator by endothelial cells; fumagillin acid derivatives which inhibit proliferation of endothelial cells and tube-formation; polysaccharide sulfate SD-4152 which inhibits proliferation and migration of endothelial cells; and retinoic acid which is responsible for modification of endothelial cell differentiation [Tsutomu Oikawa, Kekkan to Naihi, vol. 2, pp. 470-480 (1992)].
However, the above-mentioned drugs which inhibit angiogenesis have not been complete therapeutic agents for clinically suppressing angiogenesis, since some of them cause strong side-effects, thereby posing problems in terms of safety, and others only show insufficient effects.
It is therefore an object of the present invention to provide a pharmaceutical agent which provides strong angiogenesis-inhibitory effects.
According to the present invention, there has been provided (1) an angiogenesis inhibitor comprising a cysteine protease inhibitory compound.
Cysteine protease is a protease having a cysteine residue in the active site of the enzyme molecule and includes such species as cathepsin B, H, and L and dipeptidyl peptidase, all of which are lysosomal enzyme fractions, and calpain which occurs in the cytoplasm, among others. Though much remains to be explored about the physiological roles of these enzymes, a considerable amount of light has been cast on their roles in recent years. For example, calpain is known to be a protease ubiquitous in life, which is activated by calcium ions and has the optimum pH in neutral. As elucidated to this day, it takes part in degradation of the skeletal protein of cells, activation of inert cell precursors such as protein kinase C, and degradation of receptor proteins. It has also been shown that the abnormality of this enzyme activity is involved in many diseases. For example, its involvement in refractory diseases such as cerebral apoplexy (stroke), subarachnoid hemorrhage, Alzheimer""s disease, ischemic diseases, muscular dystrophy, cataract, platelet aggregation disorder, arthritis, and osteoporosis, among other diseases. [Trends in Pharmacological Science, Vol. 15, p. 412 (1994)].
The angiogenesis inhibitor of the present invention may have the following modes.
(2) The angiogenesis inhibitor of above (1) wherein the cysteine protease inhibitory compound is a calpain inhibitory compound.
(3) The angiogenesis inhibitor of above (2) wherein the calpain inhibitory compound is at least one compound selected from calpastatin and calpastatin peptide.
(4) The angiogenesis inhibitor of above (3) wherein the calpastatin peptide is at least one compound selected from peptides having an amino acid sequence of the following formula:
-Gly-A-Tyr-Arg-
xe2x80x83wherein A is -Lys-Arg-Glu-Val-Thr-Ile-Pro-Pro-Lys- (SEQ ID NO.1), -Lys-Arg-Glu-Val-Thr-Leu-Pro-Pro-Lys- (SEQ ID NO.2), -Glu-Asp-Asp-Glu-Thr-Ile-Pro-Ser-Glu- (SEQ ID NO.3), -Glu-Asp-Asp-Glu-Thr-Val-Pro-Pro-Glu- (SEQ ID NO.4), -Glu-Asp-Asp-Glu-Thr-Val-Pro-Ala-Glu- (SEQ ID NO.5), -Glu-Lys-Glu-Glu-Thr-Ile-Pro-Pro-Asp- (SEQ ID NO.6) or -Glu-Arg-Asp-Asp-Thr-Ile-Pro-Pro-Glu- (SEQ ID NO.7).
(5) The angiogenesis inhibitor of above (4) wherein the calpastatin peptide has an amino acid sequence of the following formula:
Asp-Pro-Met-Ser-Ser-Thr-Tyr-Ile-Glu-Glu-Leu-Gly-Lys-Arg-Glu-Val-Thr-Ile-Pro-Pro-Lys-Tyr-Arg-Glu-Leu-Leu-Ala.xe2x80x83xe2x80x83(SEQ ID NO.8).
(6) The angiogenesis inhibitor of above (2) wherein the calpain inhibitory compound inhibits Ca2+-binding site having a high homology with calmodulin in calpain.
(7) The angiogenesis inhibitor of above (6) wherein the compound which inhibits Ca2+-binding site having a high homology with calmodulin is at least one compound selected from calmodulin antagonistic compounds.
(8) The angiogenesis inhibitor of above (1) wherein the cysteine protease inhibitory compound is at least one compound selected from the group consisting of epoxysuccinic peptide compounds, peptide aldehyde compounds, peptide halomethane compounds, peptide diazomethane compounds, peptide halohydrazide compounds, peptide disulfide compounds, peptide ketoamide compounds and isocoumarine compounds.
(9) The angiogenesis inhibitor of above (8) wherein the cysteine protease inhibitory compound is an epoxysuccinic peptide compound.
(10) The angiogenesis inhibitor of above (9) wherein the cysteine protease inhibitory compound is an epoxysuccinic peptide compound of the formula (I): 
xe2x80x83wherein
R1 is an optionally esterified carboxy or an optionally substituted carboxamide;
R2 is a hydrogen or a lower (unless otherwise specified, xe2x80x9clowerxe2x80x9d means xe2x80x9chaving 1 to 6 carbon atomsxe2x80x9d in the present specification) alkyl or forms a ring together with R3 or R4;
R3 and R4 are the same or different and each is a hydrogen, an optionally substituted lower alkyl, an optionally substituted sulfide, or R3 and R4 combindly form a ring;
R5 is a substituted phenyl of the formula (II) 
xe2x80x83wherein
R6 is halogen atom or alkoxy, or a substituted sulfonyl of the formula (III)
SO2xe2x80x94R7xe2x80x83xe2x80x83(III)
xe2x80x83wherein R7 is aryl optionally substituted by lower alkyl or optionally substituted amino; and
n is 0 or 1,
or a salt thereof.
(11) The angiogenesis inhibitor of above (10) wherein R1 is an optionally esterified carboxy, or carboxamide optionally substituted by hydroxy or aralkyloxy.
(12) The angiogenesis inhibitor of above (10) wherein R2 is hydrogen or methyl.
(13) The angiogenesis inhibitor of above (10) wherein R2 and R3, or R2 and R4 combinedly form a pyrrolidine ring.
(14) The angiogenesis inhibitor of above (10) wherein R3 and R4 are the same or different and each is hydrogen, lower alkyl optionally substituted by aromatic group or carbamoyl, or sulfide optionally substituted by acylamino.
(15) The angiogenesis inhibitor of above (10) wherein R3 and R4 combinedly form a cyclopentane ring.
(16) The angiogenesis inhibitor of above (10) wherein R6 of the formula (II) is chlorine or fluorine.
(17) The angiogenesis inhibitor of above (10) wherein R7 of the formula (III) is phenyl or dimethylamino optionally substituted by lower alkyl.
(18) The angiogenesis inhibitor of above (8) wherein the cysteine protease inhibitory compound is a peptide aldehyde compound.
(19) The angiogenesis inhibitor of above (18) wherein the peptide aldehyde compound is leupeptin.
(20) The angiogenesis inhibitor of above (18) wherein the peptide aldehyde compound is a compound of the formula (VI): 
xe2x80x83wherein R11 is an optionally substituted aryl having 6 to 10 carbon atoms; R12 and R13 are the same or different and each is a hydrogen, a C1-C4 is alkyl, or R12 and R13 combinedly form a ring having 3 to 7 carbon atoms; and R14 is a lower alkyl optionally substituted by aryl, cycloalkyl or aromatic heterocycle, or a salt thereof.
(21) The angiogenesis inhibitor of above (20) wherein R11 is phenyl or naphthyl optionally substituted by fluorine, chlorine or methyl.
(22) The angiogenesis inhibitor of above (21) wherein R11 is a member selected from 4-fluorophenyl, 4-chlorophenyl, p-tolyl and 2-naphthyl.
(23) The angiogenesis inhibitor of above (20) wherein R12 is propyl, isopropyl or tert-butyl, and R13 is hydrogen.
(24) The angiogenesis inhibitor of above (23) wherein R12 is isopropyl and R13 is hydrogen.
(25) The angiogenesis inhibitor of above (20) wherein R12 and R 13 combinedly form cyclohexylidene.
(26) The angiogenesis inhibitor of above (20) wherein R14 is isobutyl, benzyl, cyclohexylmethyl or indol-3-ylmethyl.